Cholinoceptive properties of human primordial, preantral, and antral oocytes: In situ hybridization and biochemical evidence for expression of cholinesterase genes

  • Gustavo Malinger
  • Haim Zakut
  • Hermona Soreq


In addition to their well-known involvement in neuromuscular junctions and in brain cholinergic synapses, cholinergic mechanisms have been implicated in the growth and maturation of oocytes in various species. Functional acetylcholine receptors were electrophysiologically demonstrated in amphibian and mammalian oocyte membranes, and activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE), was biochemically measured in the exceptionally big oocytes of the frogXenopus laevis. However, biochemical methods could not reveal whether AChE was produced within the oocytes themselves or in the surrounding follicle cells. Furthermore, this issue is particularly important for understanding growth and fertilization processes in the much smaller human oocytes, in which the sensitivity of AChE biochemical measurements is far too low to be employed. To resolve this question, a molecular biology approach was combined with biochemical measurements on ovarian extracts and sections. To directly determine whether the human cholinesterase (ChE) genes are transcriptionally active in oocytes, and, if so, at what stages in their development, the presence of ChE mRNA was pursued. For this purpose frozen ovarian sections were subjected to in situ hybridization using35S-labeled human ChE cDNA. Highly pronounced hybridization signals were localized within oocytes in primordial, preantral, and antral follicles, but not in other ovarian cell types, demonstrating that within the human ovary ChE mRNA is selectively synthesized in viable oocytes at different developmental stages. Sucrose gradient centrifugation followed by [3H]acetylcholine hydrolysis measurements revealed in the ovarian extracts the presence of low levels of soluble AChE dimers, sensitive to the specific AChE inhibitor BW284C51 but resistant to the BuChE inhibitor iso-OMPA. In view of the low numbers of oocytes out of total cells in the ovary, these findings strongly suggest that AChE is a prominent protein in human oocytes throughout their development and further support the hypothesis that cholinergic mechanisms may be involved in oocyte growth and maturation in humans.


Cholinesterase Xenopus Oocyte Antral Follicle Human Oocyte Oocyte Growth 


  1. Austin, L., Berry, W.K. (1953). Two selective inhibitors of cholinesterase. Biochem J. 54:695–700PubMedGoogle Scholar
  2. Branks, P.L., Wilson, M.C. (1986). Patterns of gene expression in the murine brain revealed by in situ hybridization of brain-specific mRNAs. Mol. Brain Res. 1:1–6CrossRefGoogle Scholar
  3. Caratsch, C., Eusebi, F., Salustri, A. (1984). Acetylcholine receptors in monkey and rabbit oocytes. J. Cell. Physiol. 121:415–418PubMedCrossRefGoogle Scholar
  4. Cox, K.H., DeLeon, D.V., Angerer, L.M., Angerer, R.C. (1984). Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101:485–502PubMedCrossRefGoogle Scholar
  5. Dascal, N., Landau, E.M. (1980). Types of muscarinic response inXenopus oocytes. Life Sci. 27:1423–1428PubMedCrossRefGoogle Scholar
  6. Dascal, N., Yekuel, R., Oron, Y. (1984). Acetylcholine promotes progesterone-induced maturation inXenopus oocytes. J. Exp. Zool. 230:131–135PubMedCrossRefGoogle Scholar
  7. Drews, U. (1985). Cholinesterase in embryonic development. Prog. Histochem. Cytochem. 7:1–52Google Scholar
  8. Egozi, Y., Sokolovsky, M., Schejter, E., Blatt, I., Zakut, H., Matzkel, A., Soreq, H. (1986). Divergent regulation of muscarinic binding sites and of acetylcholines-terase in discrete regions of the developing human fetal brain. Cell. Mol. Neurobiol. 6:66–70CrossRefGoogle Scholar
  9. Eppig, J.J., Downs, S.M. (1984). Chemical signals that regulate mammalian oocyte maturation. Biol. Reprod. 30:1–11PubMedCrossRefGoogle Scholar
  10. Eusebi, F., Mangia, F., Alfei, I. (1979). Acetylcholine-elicted responses in primary and secondary mammalian oocytes disappear after fertilization. Nature 277:651–653PubMedCrossRefGoogle Scholar
  11. Eusebi, F., Pasetto, N., Siracusa, G. (1984). Acetylcholine receptors in human oocytes. J. Physiol. 346:321–330PubMedGoogle Scholar
  12. Flormann, H.M., Storey, B.T. (1981). Inhibition of in vitro fertilization of mouse eggs: 3-Guinciclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa. J. Exp. Zool. 216:159–167CrossRefGoogle Scholar
  13. Gundersen, C.B., Miledi, R. (1985). Acetylcholinesterase activity ofXenopus Laevis oocytes. Neuroscience 10:1487–1495CrossRefGoogle Scholar
  14. Johnson, C.D., Russell, R.L. (1975). A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64:229–238PubMedCrossRefGoogle Scholar
  15. Kusano, K., Miledi, R., Stinnakre, J. (1977). Acetylcholine receptors in the oocyte membrane. Nature 270:739–741PubMedCrossRefGoogle Scholar
  16. Layer, P.G. (1983). Comparative localization of acetylcholinesterase and pseudocholinesterase during morphogenesis of the chicken brain. Proc. Natl. Acad. Sci. U.S.A. 80:413–417CrossRefGoogle Scholar
  17. Layer, P.G., Sporns, O. (1987). Spatiotemporal relationship of embryonic cholinesterases with cell proliferation in chicken brain and eye. Proc. Natl. Acad. Sci. U.S.A. 84:284–288PubMedCrossRefGoogle Scholar
  18. Levanon, D., Lieman-Hurwitz, J., Dafni, N., Wigderson, M., Sherman, L., Bernstein, Y., Laver-Rudich, Z., Danciger, E., Stein, O., Groner, Y. (1985). Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the CuZn-superoxide dismutase. EMBO J. 4:77–84PubMedGoogle Scholar
  19. Lockridge, O., LaDu, B. (1986). Amino acid sequence of the active site of human serum ChE from usual, atypical and atypical-silent genotypes. Biochem. Genetics 24:485–498CrossRefGoogle Scholar
  20. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982). Molecular Cloning and Laboratory Mannual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New YorkGoogle Scholar
  21. Massoulie, J., Bon, S. (1982). The molecular forms of ChE and AchE in vertebrates. Annu. Rev. Neurosci. 5:57–106PubMedCrossRefGoogle Scholar
  22. Oron, Y., Dascal, N., Nadler, E., Lupu, M. (1985). Inositol 1,4,5-triphosphate mimics muscarinic response inXenopus oocytes. Nature 313:141–143PubMedCrossRefGoogle Scholar
  23. Owman, Ch., Sjoberg, N.O., Svensson, K.G., Walles, B. (1975). Autonomic nerves mediating contractility in the human graafian follicle. J. Reprod. Fertil 45:553–566PubMedGoogle Scholar
  24. Picard, A., Giraud, F., Le Bouffant, F., Sladeczek, F., Le Pench, C., and Doree, M. (1985). Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes. FEBS Lett. 182:446–450PubMedCrossRefGoogle Scholar
  25. Prody, L., Zevin-Sonkin, D., Gnatt, A., Koch, R., Zisling, R., Goldberg, O., Soreq, H. (1986). Use of synthetic oligodeoxynucleotide probes for the isolation of a human ChEcDNA clone. J. Neurosci. Res. 16:25–35PubMedCrossRefGoogle Scholar
  26. Prody, C., Zevin-Sonkin, D., Gnatt, A., Goldberg, O., Soreq, H. (1987). Isolation and characterization of full length cDNA clones coding for cholinesterase from fetal human tissues. Proc. Natl. Acad. Sci. U.S.A. 84:3555–3559PubMedCrossRefGoogle Scholar
  27. Razon, N., Soreq, H., Roth, E., Bartal, A., Silman, I. (1984). Characterization of levels and forms of cholinesterases in human primary brain tumors. Exp. Neurol. 84:681–695PubMedCrossRefGoogle Scholar
  28. Sastry, B.V.R., Sadavongvivad, C. (1979). Cholinergic systems in non-nervous tissues. Pharmacol. Rev. 39:65–132Google Scholar
  29. Soreq, H. (1985). The biosynthesis of biologically active proteins in mRNA-microinjectedXenopus oocytes: CRC Crit. Rev. Biochem. 18:199–238CrossRefGoogle Scholar
  30. Soreq, H., Gnatt, A. (1987). Molecular biological search for human genes encoding cholinesterases. Mol. Neurobiol. 1:47–80PubMedCrossRefGoogle Scholar
  31. Soreq, H., Miskin, R. (1984). Secreted proteins in the medium and microinjectedXenopus oocytes are degraded by oocyte proteases. FEBS Lett. 128:305–310CrossRefGoogle Scholar
  32. Soreq, H., Parvari, R., Silman, I. (1982). Biosynthesis and secretion of catalytically active acetylcholinesterase inXenopus oocytes microinjected with mRNA from rat brain and from torpedo electric organ. Proc. Natl. Acad. Sci. U.S.A. 79:830–835PubMedCrossRefGoogle Scholar
  33. Soreq, H., Zevin-Sonkin, D., Razon, N. (1984). Expression of cholinesterase gene(s) in human brain tissue: Translational evidence for multiple mRNA species. EMBO J. 3:1371–1375PubMedGoogle Scholar
  34. Soreq, H., Zevin-Sonkin, D., Avni, A., Hall, L.M.C., Spierer, P. (1985). A human acetylcholinesterase gene identified by homology to theDrosophila gene. Proc. Natl. Acad. Sci. U.S.A. 82:1827–1831PubMedCrossRefGoogle Scholar
  35. Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503–517PubMedCrossRefGoogle Scholar
  36. Woodland, H.R., Wilt, F.H. (1980). The functional stability of sea urchin histone mRNA injected into oocytes ofXenopus laevis. Dev. Biol. 75:199–204PubMedCrossRefGoogle Scholar
  37. Wramsby, H., Fredga, K., Liedholm, P. (1987). Chromosome analysis of human oocytes recovered from preovulatory follicles in stimulated cycles. N. Engl. J. Med. 316:121–124PubMedGoogle Scholar
  38. Zakut, H., Matzkel, A., Schejter, E., Avni, A., Soreq, H. (1985). Polymorphism of acetylcholinesterase in discrete regions of the developing human fetal brain. J. Neurochem. 45:382–389PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1989

Authors and Affiliations

  • Gustavo Malinger
    • 1
  • Haim Zakut
    • 1
  • Hermona Soreq
    • 2
  1. 1.Department of Obstetrics and Gynecology, The Edith Wolfson Medical Center, Holon, The Sackler Faculty of MedicineTel-Aviv UniversityIsrael
  2. 2.Department of Biology Chemistry, Institute of Life SciencesThe Hebrew UniversityJerusalemIsrael

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